Activation of death receptors offers a possible mechanism of bypassing sites of resistance and engaging the caspase machinery of apoptosis directly Death receptor pathways also cooperate in unknown ways with other signals to amplify apoptosis. For several highly resistant mesothelioma lines, including M28, the TNF-related apoptosis inducing ligand (TRAIL) induces apoptosis, an apoptosis greatly amplified by concurrent treatment either with chemotherapeutic agents, such as etoposide, or with proteasome inhibitors In our proposal, the mechanism(s) by which TRAIL-induced apoptosis can be amplified and the relevance of these findings to in vivo models of mesothelioma will be explored. First, specific pathways by which etoposide amplifies TRAIL-induced apoptosis will be investigated, with particular attention to the role of mitochondria, by blocking mitochondrial pathways with overexpression of Bcl-XL or dominant negative caspase 9, by testing mitochondrial sensitivity to the death receptor signal tBid, and by analysis of mitochondrial BH3-containing proteins. The possible role of the stress activated pathway, JNK/SAPK, will be examined in M28 cells with stable blockade with dominant negative JNK1 and/or JNK2 or with stimulation of JNK by MEKK4 Secondly, the mechanism(s) by which proteasome inhibitors amplify TRAIL-induced apoptosis will be investigated Proteasome inhibitors, unlike chemotherapeutic agents, increase expression of the TRAIL receptor, DR5. This suggests a novel role for the proteasome in the degradation and regulation of expression of a death receptor. The role of the proteasome in DR5 degradation, the specificity for DR5 and the contribution of increased DR5 to the amplified apoptosis will be determined. Finally, the in vitro findings will be tested lot their relevance to in vivo mesothelioma models In nude mice with subcutaneous tumors of M28 cells, systemic TRAIL and either chemotherapy, proteasome inhibitors or both given systemically will be assessed for effects on tumor size, apoptotic index and expression of the DR5 receptor In human mesothelioma tumors from individual patients studied as tumor fragment spheroids, response to TRAIL and/or etoposide, proteasome inhibitors or both will also be determined Understanding the mechanisms of amplification of TRAIL-induced apoptosis can provide insight into the means by which malignant cells evade apoptosis and ways to bypass those sites of resistance and thereby improve treatment strategies